Project Summary This study seeks to understand the molecular events that trigger the chronic inflammatory response during HIV infection and to better define how drugs of abuse enhance this response. We hypothesize that chronic inflammation is initiated by the pyroptotic death of resting CD4 T cells abortively infected with HIV. Pyroptosis is a highly inflammatory form of programmed cell death involving inflammasome assembly, caspase-1 activation, and membrane pore formation. Pyroptosis uniquely unites the two pathologic hallmarks of HIV infection??CD4 T cell depletion and chronic inflammation??in a single process. Pyroptosis also likely promotes inflammation-sustaining microbial translocation through damaging effects on the gut epithelium. Drugs of abuse like cocaine and opioids are known to increase the cellular expression of inflammasome-associated proteins including NLRP3 and pro-IL-1??thus potentially priming CD4 T cells for heightened levels of pyroptosis. Further, these drugs up-regulate the expression of the CCL2 and CCL7 chemokines that recruit monocytes, memory CD4 T cells, and dendritic cells to the sites of pyroptosis thereby ?adding cellular fuel to the fire.? The fact that central memory CD4 T cells displaying CCR5, CCR2, and ?the ?4?7 integrin are recruited raises the intriguing possibility that initial seeding of the latent HIV reservoir occurs within zones of pyroptotic inflammation. Pyroptosis also persists in some subjects receiving antiretroviral therapy (ART) likely because this death pathway is self-amplifying through the release of ATP, an inducer of pyroptosis. From a therapeutic perspective, these various caspase-1 dependent inflammatory effects can be blocked with VX-765, a caspase-1 inhibitor already found safe and well tolerated in humans. To assess the extent to which pyroptosis is occurring HIV-infected drug abusers, we propose to measure levels of caspase-1 activation and other pyroptotic markers occurring in CD4 T cells isolated from HIV-infected patients using cocaine or opiates versus HIV-infected subjects not using these drugs. We will also assess plasma from these individuals for the presence of cleaved IL-18 and IL-1? plus other soluble inflammatory markers. Patients will be obtained through the UCSF SCOPE cohort (Aim 1). In parallel, humanized mice will be implanted with hematopoietic stem cells (HSCs) containing or lacking the CASP1 gene and infected with R5 transmitted-founder viruses. Recombinant Cas9 and caspase-1 specific guide RNAs will be nucleofected into HSCs to knockout CASP1 expression. These mice will be studied for levels of inflammation (and CD4 T cell depletion) following infection and studied to assess whether CASP1 deficiency alters inflammation induced by cocaine or opioids (Aim2). Finally, this mouse model will also be used to investigate whether pyroptotic inflammation propels initial seeding of the latent HIV reservoir (Aim 3). Through the use of different experimental approaches, we will explore caspase-1 driven pyroptosis as an initiator and driver of chronic inflammation in HIV infection and determine whether drugs of abuse enhance this signaling pathway.